The present invention relates to a device and method for performing a biological modification of a fluid and, more particularly, to an artificial installation, which supplements, augments or replaces organ function. Specifically, the artificial installation may contain liver micro-organ cultures. Further specifically, the artificial installation may contain a viable kidney micro-organ component. Further specifically, the artificial installation may contain a dialysis component as commonly used in hemodialysis. The kidney micro-organ culture and dialysis unit together perform physiologically as a kidney substitute.
Further, the present invention relates to a device and method for supplementing, augmenting or replacing both hepatic and renal function via use of a single artificial installation containing both liver and kidney micro-organ cultures.
Further, the present invention relates to a device and method for supplementing, augmenting or replacing both hepatic and renal function via use of a single artificial installation containing both liver and kidney micro-organ cultures in a ratio of approximately 6:1.
Further, the present invention relates to an artificial installation containing a combined liver and kidney micro-organ culture additionally containing a dialysis component.
Further, the present invention relates to a method of preparing viable tissue which can be stored in an artificial installation for supplementing, augmenting or replacing organ function without culturing the tissue prior to storage and for transplantation at a later stage into a host.
A number of organs in the body, such as the liver and kidney, modify body fluids such as blood. The kidney is a multifunctional organ, excreting nitrogenous waste in the form of urea, excreting excess inorganic salts, and actively secreting erythropoietin and other substances, such as, but not limited to rennin and tissue plasminogen activator. The liver removes toxic substances from the blood and performs many biochemical functions such as, but not limited to, detoxifying ammonia into urea, bilirubin metabolism, glycogen storage, lipid synthesis, drug metabolism, albumin secretion and clotting factor secretion. Hepatic and renal functions are closely related, with many metabolic byproducts and toxins passing from the liver, via the circulatory system, to the kidney for excretion into the urine. Thus, the liver and kidney are essential organs, and the synergy between these two organs is crucial. Patients with liver or kidney failure are at high risk for mortality if immediate intervention is not effected.
There are many causes of liver failure, including, for example, exposure to toxic substances, hepatitis, and genetic defects (Kasai, et al., Artif. Organs, 18:348-54, 1994). Currently, 70% of patients with acute liver failure die because of no available treatment (Kasai, et al., Artif. Organs, 18:348-54, 1994). Furthermore, 10-30% of patients die while awaiting donor liver organs (LePage, et al., Am. J. Crit. Care, 3:224-7, 1994; Sussman, et al. Artif. Organs, 18:390-6, 1994; and Uchino and Matsushita, Asaio J., 40:74-7, 1994).
A bedside life-support device that could temporarily perform liver function during liver failure is called an Extracorporeal Liver Devicexe2x80x94ELD. The development and commercialization of such a device would clearly be of enormous benefit for a number of reasons (Fox et al, Am. J. Gastroenterol., 88:1876-81, 1993). An ELD would benefit the roughly 2,000 patients with fulminant liver failure (FH) in the U.S. each year (Hoofnagle, et al., Hepatology, 21:240-52, 1995). It could also be used as a bridge to liver transplantation for patients awaiting donor organs.
An ELD that would function for several weeks could in addition allow for recovery to normal functioning of the patient""s own liver. Since it is unlikely that every hepatocyte is destroyed in a damaged liver, adequate liver support for two to three weeks could allow surviving hepatocytes to repopulate the damaged liver. Fewer than a dozen hepatocytes are required to repopulate the liver in an animal model of lethal hepatic disease Sandgren et al., Cell, 66:245-56, 1991). A patient with 90-95% liver necrosis should be able to recover sufficient function to survive independently after only a few days of support (Sussman et al. Artif. Organs, 18:390-6, 1994).
In an attempt to provide such an ELD, several purely mechanical, non-biological blood-treatment devices have been developed. In the most basic form, the purpose of these devices is to selectively remove toxins and add nutrients across a membrane with a relatively small pore size. One of the most advanced of these non-biological devices has been developed by Hemocleanse(trademark) and has recently received FDA approval. In a randomized, controlled clinical trial using the Hemocleanse(trademark) apparatus, removal of metabolites was limited and there was no significant effect on blood ammonia levels (Hughes et al., Int. J. Artif. Org., 17:657-662, 1994). Clearly, liver function is extremely complex and is unlikely to be replaced by a solely mechanical or a chemical device at this time.
Other currently available ELDs use biological materials as a starting point. For example, one of the most clinically tested device, called ELAD (for extracorporeal liver assist device) uses a transformed immortalized human cell line as a source for hepatocyte-like cells (Sussman, et al. Artif. Organs, 18:390-6, 1994). Initial trials of this device were performed under xe2x80x9cEmergency Use of Unapproved Medical Devicesxe2x80x9d, or xe2x80x9cInvestigational Device Exemptionxe2x80x9d. Efficacy was not determined, but no serious adverse side effects were observed except for clotting that was managed by drug treatment. While the use of an immortalized human cell line is convenient because it provides an expendable source of cells, there are two major reasons why it may not be ideal. Firstly, there are obvious safety and regulatory concerns about using immortalized cell lines in clinical practice. Secondly, immortalized cells would not be expected to rein all the normal physiological characteristics of primary hepatocytes, particularly after industrial scale expansion (Sussman et al., Artif Organs, 18:90-6, 1994).
A second general approach for obtaining liver cells as a source for an ELD, is the isolation of liver cells or tissue from intact livers. In previous attempts, cells from livers have usually been disassociated using enzymes such as collagenase, which disrupts the normal micro architecture of the liver. Some attempts have been used to use liver pieces, but the shape of these pieces have not been designed for proper surface area to volume ratios necessary for optimal tissue maintenance (Lie et al., Res Exp Med (Berl) 185:483-94, 1985).
One current limitation is the ability of current methods of culturing mammalian liver cells to provide conditions which allow cells to assemble into tissues which simulate the spatial three-dimensional form of actual tissues in the intact organism. Conventional tissue culture processes limit, for similar reasons, the capacity for cultured tissues to express a highly functionally specialized or different state?? considered crucial for mammalian cell differentiation and secretion of specialized biologically active molecules of research and pharmaceutical interest. Unlike microorganisms, the cells of higher organisms such as mammals form themselves into high order multi-cellular tissues. Although the exact mechanisms of this self-assembly are not known, in the cases that have been stated so far, development of cells into tissues has been found to be dependent on orientation of the cells with respect to each other or another anchorage substrate and/or the presence or absence of certain substances such as hormones. In summary, no conventional culture process used in the organ assist devices to date is capable of simultaneously achieving proper functioning of the cells in vitro while at the same time maintaining critical cell/cell/substrate interactions and proper micro-environment to allow excellent modeling of in vivo organ tissue structure and function. The fact that the present invention provides a method for using organ tissue, including liver tissue, in an ELD is an important advancement relative to prior art teachings.
This method for using organ tissue, including but not limited to, liver tissue, can rely on cryo-preservation prior or following micro-organ culturing. Components of a suitable cryo-preservation solution might include, but are not limited to, raffinose or trehalose as taught by U.S. Pat. No. 5,879,875 which is fully incorporated herein by reference.
In the liver, the unique juxtaposition of diverse cell populations and matrix components in harmony with the angio-architecture results in a delicate bioecological system. It is therefore unlikely that standard cultures of hepatocytes will perform even the minimal liver functions. As mentioned previously, the cells of higher organisms such as mammals form themselves into high order multi-cellular tissues. An example of physical contact between a cell and a non cellular substrate (matrix) is the physical contact between an epithelial cell and its basal lamina. Examples of functional contact between one cell and another cell include electrical or chemical communication between cells. For example, cardiomyocytes communicate with other cardiomyocytes via electrical impulses. In addition, many cells communicate with other cells via chemical messages, e.g., hormones, which either diffuse locally (paracrine signaling and autocrine signaling), or are transported by the vascular system to more remote locations (endocrine signaling). Examples of paracrine signaling between cells are the messages produced by various cells (known as enteroendocrine cells) of the digestive tract, e.g., pyloric D cells which secrete somatostatin which in turn inhibits the release of gastrin by nearby pyloric gastrin (G) cells. This micro-architecture can be extremely important for the maintenance of a tissue explant of an organ in minimal media, e.g., without exogenous sources of serum or growth factors, because the liver tissue can be sustained in such minimal media by paracrine and autocrine factors resulting from specific cellular interactions within the micro-organ.
The preparation of such a micro-organ culture is described in U.S. Pat. No. 5,888,720 and U.S. patent application Ser. No. 08/482,364, both herein incorporated by reference. In the preparation of a micro-organ culture, the populations of cells which make up the explant are isolated from a liver in a manner that preserves the natural affinity of one cell to another, e.g., to preserve layers of different cells as present in the organ itself. For example, in skin micro-organ cultures, keratinocytes of the epidermis remain associated with the stroma and the normal tissue architecture is preserved including the hair follicles and glands. This basic structure is common to all organs, for instance, which contain an epithelial and a stromal component. Moreover, such an association facilitates intercellular communication. This is particularly important in differentiating cells where induction is defined as the interaction between one (inducing) and another (responding) tissue or cell, as a result of which the responding cells undergo a change in the direction of differentiation. Moreover, inductive interactions occur in embryonic and adult cells and can act to establish and maintain morphogenetic patterns as well as induce differentiation (Gurdon, Cell, 68:185-199, 1992).
Furthermore, the micro-organ cultures prepared according to U.S. patent application Ser. No. 08/482,364 preserve normal liver tissue architecture even when cultured for prolonged periods of time. Because these cultures can be maintained in controlled and uniform conditions and yet closely resemble tissue in vivo, they provide a unique continuous source of functional liver cells in vitro.
None of the prior art organ assist devices, or related devices in the prior art, uses micro-organ cultures or cryo-micro-organs in order to perform a biological modification of a fluid.
In the United States, approximately 200,000 people develop acute renal failure (ARF). The mortality rate for this group is approximately 50%. Treatment is limited to supportive care, dialysis and transplantation. Even the continuous dialysis often used in these situations is inadequate. For most patients, timely transplantation is not an option. The National Kidney Foundation estimates there are 53,000 Americans waiting for life-saving transplants; 10 people die each day while waiting.
Another major health care need is in supplementing or replacing kidney dialysis. Unfortunately, current dialysis methods represent a clinically poor and economically expensive solution to kidney failure. Dialysis attempts to replace a highly sophisticated mechanical and regulatory organ with a fairly simple filtering system.
About 250,000 people are treated by dialysis in the United States at a cost of billions of dollars. The ESRD population is growing by 7%-9% per year; by 2010, there will be more than 350,000 such patients. The average cost of providing care for a patient receiving dialysis is US $45,000 per year.
While patients have a more flexible life style with a transplant, and experience greater well being, as noted above, the waiting list for kidney transplants is approximately 50,000. Only about a fifth of those waiting received transplants last year.
Hemodialysis as a mode of treatment has several inherent disadvantages. First, it requires the use of expensive equipment and skilled paramedical personnel to operate the equipment. Second, since treatments are generally conducted several times per week on an outpatient basis, costs are high and patients are required to remain close to their treatment facility. In addition, patients undergoing treatment must remain on a strict diet between treatment sessions, often leading to medical crises due to poor compliance. Each round of treatment lasts several hours and requires venous puncture for connection to the hemodialysis machine. As a result, some long term hemodialysis patients suffer venous collapse in the most easily accessible veins of the arm.
Kidney transplantation is considered the preferred mode of treatment for patients lacking kidney function, although it has its own set of inherent drawbacks. While routinely performed in many medical centers, it still carries with it all the risks associated with intra-abdominal surgery. In addition, transplant recipients are often treated with immuno-suppressant drugs for a prolonged period after surgery to prevent organ rejection, a practice which places them at increased risk of infection. Most problematic is the chronic shortage of available kidneys for transplantation, and the difficulty in matching donated organs to suitable recipients. Even in the case of a successful transplant, the recipient has only one kidney. Supplementation of the function of a transplanted kidney with a device such as that described in the present invention could increase this post transplantation survival time by reducing the load on the transplanted kidney.
In an attempt to address the chronic shortage of donated kidneys, considerable effort has been directed towards development of genetically engineered humanized pigs to serve as organ donors. Unfortunately, the recent discovery of porcine endogenous retroviruses (pervs) has raised serious doubts about the advisability of xenotransplantation even if it becomes technically feasible. Use of micro-organ cultures isolated physically from the patient, as described, could serve as an enabling technology which would facilitate safe use of these modified porcine organs in humans.
There is thus a widely recognized need for, and it would be highly advantageous to have, a device and method for supplementing, augmenting or replacing liver function, kidney function, or both liver and kidney function, devoid of the above limitations. Such a device and method would allow patients to have a better quality of life while awaiting transplantation and also serve to increase their life expectancy after receipt of a new organ and in some instances to allow for their own organ to recover while being supported by the device.
U.S. Pat. No. 5,888,720 teaches an in vitro micro-organ culture system, and a method for preparing same, for growth of a population of non-fetal animal cells in which epithelial and stromal cells are cultured together in a nutrient medium and remain viable for more than 24 hours. More preferably that patent relates to an in vitro micro-organ culture system in which the population is composed of an epithelium and a stroma which are cultured together and maintain the stromal and epithelial tissues, as determined by histology, while remaining viable as determined by DNA synthesis for at least 48 hours in the absence of serum. Most preferably that patent relates to an in vitro micro-organ culture system in which epithelial and stromal cells are cultured together and maintain the stromal and epithelial tissues, as determined by histology, while remaining viable as determined by DNA synthesis for at least seven days in the absence of serum. According to the teachings of U.S. Pat. No. 5,888,720, these micro-organ cultures are devoid of an internally disposed synthetic support structure and absent a sandwich support structure.
U.S. Pat. No. 5,888,720 further teaches that the first dimension of the micro-organ culture is not greater than the second dimension and smaller than the third dimension, the first dimension being measured in a direction that is substantially parallel with the exterior surface of the organ from which the micro-organ culture is derived. That patent further teaches that the non-fetal animal cells disposed in the nutrient medium are derived from an organ with an in vivo tissue structure including an epithelial tissue and an adjacent stroma, wherein the epithelial tissue has a first surface corresponding to an exterior surface of the organ and a second surface in contact with the stroma. Such a micro-organ culture could be derived from organs including, but not limited to, lung, duodenum, esophagus, intestine, colon, liver and pancreas.
According to U.S. Pat. No. 5,888,720, the surface area to volume index of the micro-organ culture is defined as xe2x80x9cAlephxe2x80x9d where Aleph=1/x+1/a greater than 1.5 mmxe2x88x921 and x=tissue thickness and a=width of tissue in millimeters. The patent teaches that the first dimension of the micro-organ culture is not greater than the second dimension and smaller than the third dimension wherein the first dimension is no greater than 0.45 mm. The third dimension is ignored in determining the surface area to volume index because variation in the third dimension causes ratiometric in both volume and surface area. Therefore, when determining Aleph, a and x should be defined as the two smallest dimensions of the tissue slice.
This surface area to volume index is a unique aspect of the invention because it allows a similar availability of nutrients to all cells in the tissue by diffusion. The diffusion of nutrients to every cell in a three dimensional micro-organ culture requires that Aleph is at least approximately 1.5 mmxe2x88x921.
Further according to the teachings of U.S. Pat. No. 5,888,720, populations of cells are grouped in a manner that preserves the natural affinity of one cell to another so that in vivo epithelial and stromal tissue architecture is preserved. For example, in skin micro-organ cultures, keratinocytes of the epidermis remain associated with the stroma and the normal tissue architecture is preserved including the hair follicles. Such an association facilitates intercellular communication. Many types of communication takes place among animal cells. This is particularly important in differentiating cells where induction is defined as the interaction between one (inducing) and another (responding) tissue or cell, as a result of which the responding cells undergo a change in the direction of differentiation. Moreover, inductive interactions occur in embryonic and adult cells and can act to establish and maintain morphogenetic patterns as well as induce differentiation (Gurdon (1992) Cell 68: 185-199).
According to the teachings of U.S. Pat. No. 5,888,720, the micro-organ cultures prepared according to the invention as described in Example 1 thereof, comprise a population of cells grouped in a manner which includes a stromal and epidermal layers so as to preserve the natural tissue architecture. This patent teaches preparation of micro-organ cultures from animals including adult human skin, mouse, guinea pig and rat skin have been isolated and grown for up to 21 days in culture. However, it is within the scope of the invention described in this patent to maintain cultures for extended periods of time beyond 21 days and to derive these cultures from other animals. Further, micro-organ cultures from skin and organs including the mammalian, pancreas, liver, kidney, duodenum and esophagus are taught. Similarly, micro-organ cultures of epithelia from mammalian cornea, kidney, breast tissue and various gut derived tissues in addition to the esophagus such as intestine and colon may also be prepared using the methods taught by U.S. Pat. No. 5,888,720.
U.S. Pat. No. 5,888,720 further teaches that these micro-organ cultures can be maintained in a simple medium such as Dulbecco""s Minimal Essential in the absence of serum while retaining stromal and epithelial tissues as determined by histology, and cell viability as determined by DNA synthesis. The media and culture may be contained in a culture vessel with an O2 pressure not substantially greater than that in the atmosphere. Furthermore, the cultures may be grown in a media containing neither sera nor any other biological fluid and can be maintained for extended periods of time for example 48 hours to 21 days.
According to the teachings of U.S. Pat. No. 5,888,720 micro-organ cultures may be maintained in any suitable culture vessel such as a 24 or 96 well microplate and may be maintained at 37xc2x0 C. in 5% CO2. The cultures may be shaken for improved aeration, the speed of shaking being for example 12 rpm.
In addition, U.S. Pat. No. 5,888,720 teaches methods for production of micro-organ cultures with the properties described hereinabove.
According to the present invention there is provided a device for performing a biological modification of a fluid, the device comprising (a) a chamber having an inlet for intake of the fluid and an outlet for outflow of the fluid; and (b) a collection of micro-organ cultures of at least one organ for performing the biological modification of the fluid, each individual micro-organ culture of the collection including cells and having dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture (organ structure) of organ unit (e.g., acinus of liver) is maintained within each individual micro-organ culture, the collection of micro-organ cultures being located within the chamber and the collection of micro-organ cultures being in contact with at least a portion of the fluid flowing through the chamber.
According to further features of preferred embodiments of the present invention the inlet and outlet for the biological fluid take the form of tubes and the device is installed extracorporeal to the patient.
According to additional further features of preferred embodiments of the present invention the inlet and outlet for the biological fluid take the form of a semipermeable membrane and the device is installed intracorporeal to the patient in the form of an intrabodily transplantable device.
Preferably the organ(s) selected includes liver and/or kidney. Also preferably, the collection of micro-organ cultures include cells from the at least one organ, such that intercellular contacts between the cells are preserved. Most preferably, each of the collection of micro-organ cultures is characterized by an Aleph of at least about 2.6 mmxe2x88x921.
According to preferred embodiments of the present invention, the micro-organ culture is substantially encapsulated by a sheet of a biocompatible polymer, the sheet being located substantially within the chamber. Preferably, the sheet has a first dimension in a range of from about 10 cm to about 90 cm, a second dimension in a range of from about 10 cm to about 90 cm and a third dimension in a range of from about 300 micrometers to about 900 micrometers. Also preferably, a plurality of the sheets are incorporated substantially parallel in orientation within the chamber, such that fluid flows freely between the sheets.
According to another embodiment of the present invention, there is provided a device for performing a biological modification of a fluid of a subject, including: (a) a chamber having an inlet for intake of the fluid and an outlet for outflow of the fluid; (b) a collection of micro-organ cultures for performing the biological modification of the fluid, each individual micro-organ culture of the collection including cells and having dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture of organ units is maintained within each individual micro-organ culture, the collection of micro-organ cultures being located within the chamber and the collection of micro-organ cultures being in contact with at least a portion of the fluid flowing through the chamber; (c) a first tube having first and second ends, the first end for coupling to the subject for receiving the fluid from the subject, the second end for coupling to the inlet; and (d) a second tube having first and second ends, the first end for coupling to the outlet and the second end for coupling to the subject to return the fluid to the subject after the biological modification.
According to still further embodiments of the present invention, there is provided a method of performing a biological modification of a fluid from a subject, the method comprising the step of perfusing a chamber containing a collection of micro-organ cultures with the fluid from the subject, such that the collection of micro-organ cultures performs the biological modification on the fluid, wherein each individual micro-organ culture of the collection includes cells and has dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture of organ units is maintained within each individual micro-organ culture.
According to still further embodiments of the present invention, there is provided a method of preparing a continuous planar organ. The method comprising the steps of (a) obtaining a collection of individual micro-organ cultures of an organ, such that each of the individual micro-organ culture of the collection includes cells and has dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo organ architecture of organ units is maintained within each individual micro-organ culture; and (b) adding (e.g., layering) a suspension of cells from the organ onto the micro-organ cultures and co-culturing the suspension of cells in presence of the collection of micro-organ cultures, such that the continuous planar organ is formed from an admixture of cells derived from the micro-organ cultures and the cells in suspension.
According to a preferred embodiment of the present invention, the collection of liver micro-organ cultures is provided within a continuous liver planar organ formed by culturing hepatocyte an/or endothelial or other type of cells in presence of the collection of liver micro-organ cultures, such that the continuous liver planar organ is formed from an admixture of cells derived from the micro-organ cultures and the chosen cells.
According to still further embodiments of the present invention, there is provided a method of preparing a continuous liver planar organ. The method comprising the steps of (a) obtaining a collection of individual liver micro-organ cultures, such that each of the individual micro-organ culture of the collection includes liver cells and has dimensions, such that cells positioned deepest within the individual micro-organ culture are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, thereby in vivo liver architecture of acinus units is maintained within each individual micro-organ culture; and (b) adding (e.g., layering) a suspension of hepatocyte and/or endothelial or other types of cells onto the micro-organ cultures and co-culturing the suspension of cells in presence of the collection of liver micro-organ cultures, such that the continuous planar liver organ is formed from an admixture of cells derived from the micro-organ cultures and the chosen cells.
According to one aspect of the present invention, there is provided a device for providing a subject with one or more organ functions in case of impaired organ function, the device comprising a chamber containing a plurality of micro-organ cultures, such that the micro-organ cultures are in contact with at least a portion of said subject""s blood as it flows through the chamber, wherein cells positioned deepest within an individual micro-organ culture of the plurality of micro organ cultures are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of the individual micro-organ culture, so that they maintain in vivo tissue architecture within each individual micro-organ culture, while, at the same time, allowing cells positioned deepest within each individual micro-organ culture benefit of diffusional nutrition and oxygenation thereby preventing necrosis.
According to an additional aspect of the present invention, there is provided a method for providing a subject with one or more organ functions in case of impaired organ function, the method comprising the steps of: (a) providing a chamber containing a plurality of micro-organ cultures wherein cells positioned deepest within an individual micro-organ culture are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of said individual micro-organ culture, thereby maintaining in vivo tissue architecture within each individual micro-organ is culture, while, at the same time, allowing cells positioned deepest within each individual micro-organ culture benefit of diffusional nutrition and oxygenation thereby preventing necrosis; and (b) forma fluid communication between the chamber and the circulatory system of the subject, so that blood flows from the subject through the chamber and from the chamber to the subject, such that the micro-organ cultures are in contact with at least a portion of said subject""s blood as it flows through said chamber.
According to additional features of preferred embodiments of the present invention, the chamber of the present invention may be installed extracorporeally.
According to additional features of preferred embodiments of the present invention, the chamber of the present invention may be intrabodily transplantable.
According to additional features of preferred embodiments of the present invention, the present invention includes also systems for supplementing, augmenting or replacing organ function if those systems can rely upon micro-organ cultures as a substitute and become an integral part of their function.
According to additional features of preferred embodiments of the present invention, the present invention includes also methods for supplementing, augmenting or replacing organ function if those methods rely upon micro-organ cultures as an integral part of their function.
According to additional further features of the present invention, the organ function to be supplemented augmented or replaced is hepatic organ function.
According to additional further features of the present invention, the organ function to be supplemented augmented or replaced is renal organ function.
According to additional further features of the present invention, the organ function to be supplemented augmented or replaced is renal and hepatic organ function.
According to additional further features of the present invention, the organ function to be supplemented augmented or replaced includes a dialysis function for excretion of small molecules out of the body.
According to additional further features of the present invention, blood enters and leaves the chamber via one or more tubes connected thereto and also to the circulatory system of the subject.
According to additional further features of the present invention, blood enters and leaves the chamber via an outer wall of the chamber which is constructed of a bio-compatible membrane which facilitates neo-vascularization. Prior to this neo-vascularization, blood enters and leaves the chamber via diffusion.
According to further features of preferred embodiments of the present invention, MCs are further contained within an inner semipermeable bio-compatible membrane so that plasma from blood in the chamber may diffuse into the membrane and contact the MCs and so that secretions from the MCs may diffuse into blood within the chamber for subsequent return to the circulatory system of the subject.
According to further additional features of preferred embodiments of the present invention, the bio-compatible membrane is polycarbonate.
According to further features in preferred embodiments of the invention described below, cells derived from a cell suspension are co-cultured with the plurality of micro-organ cultures such that a continuous planar organ is formed.
According to additional further features in preferred embodiments of the invention described below, the cells derived from a cell suspension are liver derived cells.
According to additional further features in preferred embodiments of the invention described below, the cells derived from a cell suspension are kidney derived cells.
According to further features in preferred embodiments of the invention described below, the micro-organ cultures are characterized by being cryo-preserved and thawed before being located within chamber.
According to further features of preferred embodiments of the present invention, the micro-organ cultures are prepared from a portion of an organ which has been cryo-preserved.
According to further features of preferred embodiments of the present invention, the liver micro-organ cultures are prepared from a portion of a liver which has been cryo-preserved.
According to further features of preferred embodiments of the present invention, the kidney micro-organ cultures are prepared from a portion of a kidney which has been cryo-preserved.
According to still further embodiments of the present invention, there is provided a method of preparing cryo-micro-organs, the method comprising the steps of (a) cryo-preserving at least part of an organ; (b) cutting the cryo-preserved organ into sections while maintaining the resulting sections in a cryo-preserved state; (c) prior to use, thawing the cryo-preserved sections of the cryo-preserved organ; and (d) employing the thawed sections as micro-organs. Such cryo-micro-organs can be incorporated in a device following thawing or they could be cultured in vitro following thawing and then incorporated into the device.
According to further features of preferred embodiments of the present invention, the organ is cryo-preserved in a solution containing at least one component selected from the group consisting of DMEM, DMSO, Glycerin, trehalose, raffinose, an antibiotic, an antimycotic and glucose.
According to additional further features of preferred embodiments of the present invention, the temperature for cryo-preservation of the organ, and for sections cut from it, is between zero degrees centigrade and xe2x88x92180 degrees centigrade.
According to still additional further features of preferred embodiments of the present invention, the thickness of the sections cut from a part of an organ is between approximately 200 and approximately 400 micrometers.
According to still additional further features of preferred embodiments of the present invention, prior to thawing for use as micro-organs, the frozen sections are stored for an additional period of time or are immediately thawed and used.
According to further features of preferred embodiments of the present invention, the employment of the thawed sections as micro-organs is accomplished by (i) providing a chamber; (ii) containing a plurality of the thawed sections within the chamber; (iii) introducing into the chamber at least a portion of a subject""s blood so that the plurality of thawed sections are in contact with the at least a portion of a subject""s blood, wherein cells positioned deepest within an individual thawed section of the plurality of thawed sections are at least about 100 or 150 micrometers and not more than about 225 micrometers away from a nearest surface of each individual thawed section, thereby maintaining in vivo tissue architecture within each individual thawed section, while, at the same time, allowing cells positioned deepest within each individual thawed section of the plurality of micro organs diffusional nutrition and oxygenation and prevention of necrosis thereof; and (iv) reintroducing into the subject at least a portion of the subjects blood after it has been in contact with the thawed sections.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a device and method for augmenting, supplementing or replacing hepatic function, renal function or both hepatic and renal function in cases where those functions are compromised. In addition, the present invention helps to address the chronic shortage of donated organs for transplant and has the potential to reduce problems of tissue rejection and transfer of disease with transplanted organs. Further, the present invention increases the efficacy with which cell masses in organ assist devices function by providing methods which preserve tissue architecture and methods which eliminate the need for culture in artificial media prior to use, and in particular imposes less stress on the cells during the preparation procedures.